Therapy-enhancing glucan

ABSTRACT

A therapeutic composition for treatment of cancer in a mammal is disclosed. The composition comprises an effective amount of a glucan composition which is suitable for oral administration and for absorption through the gastrointestinal tract of the mammal, and at least one antibody for the cancer. A method of treating cancer in a mammal is also disclosed. The method comprises administering a suitable orally administered glucan and at least one antibody for the treatment of cancer to the mammal. In addition a composition for delivery of peptide, protein, RNA, DNA or plasmid comprising effective amount of a beta-glucan is disclosed.

This application is a Continuation-In-Part of International ApplicationNo. PCT/US04/23099, Filed Jul. 16, 2004, which is a Continuation-In-Partof U.S. Ser. No. 10/621,027, Filed Jul. 16, 2003, and is aContinuation-In-Part of U.S. Ser. No. 11/218,044, Filed Aug. 31, 2005,which is a Continuation of U.S. Ser. No. 10/621,027, Filed Jul. 16,2003, which is a Continuation-In-Part of International Application No.PCT/US02/01276, Filed Jan. 15, 2002, which claims the benefit of U.S.Ser. No. 60/261,911, Filed Jan. 16, 2001. The contents of the precedingapplications are hereby incorporated herein by reference in theirentireties.

Throughout this application, various references are cited. Disclosuresof these publications in their entireties are hereby incorporated byreference into this application to more fully describe the state of theart to which this invention pertains.

BACKGROUND OF THE INVENTION

Beta-glucans have been tested for tumor therapy in mice for nearly 40years (^(1,2)). Several forms of mushroom derived beta-glucans are usedclinically to treat cancer in Japan, including PSK (from Coriolusversicolor), Lentinan and Schizophyllan. In randomized trials in Japan,PSK has moderately, but significantly improved survival rates in somecancer trials: after gastrectomy (^(3,4)), colorectal surgery(^(5,6)),and esophagectomy (⁷) to remove primary tumors. Results have been lessencouraging in breast cancer (^(8,9)), and leukemia (¹⁰). Schizophyllanhas improved survival of patients with operable gastric cancer (¹¹),inoperable gastric cancer (^(12,13)), and cervical cancer (¹⁴). Again,though survival differences between groups were statisticallysignificant, these improvements were modest. While beta-glucans are notwidely used by Western oncologists, beta-glucan containing botanicalmedicines such as Reishi and maitake (¹⁵) are widely used by U.S. cancerpatients as alternative/complementary cancer therapies. These previousstudies that looked for a therapeutic effect of beta-glucan did notincorporate co-administration of therapeutic monoclonal antibodies(MoAb) as part of the protocol. When beta-glucan is administered withoutco-administration of MoAb, its tumor cytotoxic effect requires thepresence of naturally-occurring antitumor antibodies which can be quitevariable among patients and even in experimental mice.

In Europe and USA beta-glucans especially from Bakers' yeast have longbeen employed as feed additives for animals, as dietary supplement forhumans (¹⁷), in treatment of wounds (¹⁸), and as an active ingredient inskin cream formulations. The basic structural unit in beta-glucans isthe β(1→3)-linked glucosyl units. Depending upon the source and methodof isolation, beta-glucans have various degrees of branching and oflinkages in the side chains. The frequency and hinge-structure of sidechains determines its immunomodulor effect. beta-glucans of fungal andyeast origin are normally insoluble in water, but can be made solubleeither by acid hydrolysis or by derivatisation introducing chargedgroups like -phosphate, -sulphate, -amine, -carboxymethyl and so forthto the molecule (¹⁹⁻²⁰).

SUMMARY OF THE INVENTION

This invention provides a composition comprising an effective amount ofbeta-glucan capable of enhancing efficacy of antibodies and theirderivatives. In an embodiment, the antibody is a monoclonal antibody. Ina further embodiment, the antibody is an antibody against cancer.

The cancer is recognized by antibodies, and which includes but notlimited to neuroblastoma, melanoma, non-Hodgkin's lymphoma, Epstein-Barrrelated lymphoma, Hodgkin's lymphoma, retinoblastoma, small cell lungcancer, brain tumors, leukemia, epidermoid carcinoma, prostate cancer,renal cell carcinoma, transitional cell carcinoma, breast cancer,ovarian cancer, lung cancer colon cancer, liver cancer, stomach cancer,and other gastrointestinal cancers. Antibodies in this respect refers toany part of immunoglobulin molecules having specific cancer cell bindingaffinity by which they are able to exercise anti-tumor'activity.Examples are antigen binding fragments or derivatives of antibodies.

It will be recognized by one of skill in the art that the variousembodiments of the invention relating to specific methods of.treatingtumors and cancer disease states may relate within context to thetreatment of a wide number of other tumors and/or cancers notspecifically mentioned herein. Thus, it should not be construed thatembodiments described herein for the specific cancers mentioned do notapply to other cancers.

This invention further provides the above compositions and apharmaceutically acceptable carrier, thereby forming pharmaceuticalcompositions.

This invention also provides a method for treating a subject with cancercomprising administrating the above-described composition to thesubject.

This invention provides a composition comprising effective amount ofbeta-glucan capable of enhancing host immunity. In another embodiment,the immunity is against cancer.

This invention also provides a method for introducing substances intocells comprising contacting a composition comprising orally administeredbeta-glucan with said cells.

This invention further provides a method for introducing substances intoa subject comprising administering to the subject an effective amount ofthe above compositions. The substance which could be delivered orallyincludes but is not limited to peptides, proteins, RNAs, DNAs,chemotherapeutic agents, biologically active agents, and plasmids. Othersmall molecules and compounds may be used as well.

DETAILED DESCRIPTION OF THE FIGURES

FIG. 1. Barley (1→3),(1→4)-β-D-glucan plus antibody in the treatment ofmetastatic neuroblastoma in patients. MIBG scan before and aftertreatment in a patient with metastatic neuroblastoma refractory tomultiple regimens of chemotherapy. Patient received intravenous anti-GD2antibody 3F8 (10 mg/m2/day) for a total of 10 days, plus oral barleybeta-glucan over the same time period. FIG. 1A shows baseline MIBG scanof patient. Extensive osseous metastasis can be seen in the femora,fibulae, pelvis, ribs, left scapula, right clavicle, humeri, skull andspine. Heart, liver, stomach and colon uptakes are physiologic. FIG. 1Bshows MIBG scan of same patient 2 months later, following a single cycleof therapy with 3F8 plus glucan. Areas of metastases have significantlyimproved.

FIG. 2. Barley (1→3),(1→4)-β-D-glucan plus antibody in treatment ofsubcutaneous human lymphoma xenografted in SCID mice. SCID mice withestablished subcutaneous Daudi (n=9) (FIG. 2A), Hs445 (n=5) (FIG. 2B),EBV-derived LCL (n=9) (FIG. 2C) and RPMI 6666 (n=10; data not shown)xenografts were treated either with 200 ug intravenous rituximab twiceweekly for 8 doses (▪), 400 ug (1→3),(1→4)-D-β-glucan administeredorally via intragastric gavage daily for 29 days (Δ) or a combination ofrituximab and (1→3),(1→4)-D-β-glucan (x), or left untreated) (♦).Percentage tumor growth is plotted on y-axis and days after treatmentwas commenced on x-axis. Error bars represent SEM and have been shownonly for rituximab alone and combination groups. For all xenografts,only combination treatment was associated with reduction in tumorgrowth. The reduction in tumor growth per day in the group receivingbeta-glucan in addition to rituximab compared to rituximab alone was2.0% (95% CI 1.3-2.7%; p<0.0005) for Daudi, 0.8% for EBV-derived LCL(95% CI 0.4-1.2%; p<=001), 2.2% for Hs445 (95% C.I. 1.2%-3.2%;p=0.0009), and 1.8% for RPMI6666 (95% CI 1.0-2.7%; p<0.0002) xenografts.

FIG. 3. Barley (1→3),(1→4)-β-D-glucan plus antibody in treatment ofdisseminated human lymphoma xenografted in SCID mice. 5×10⁶ Daudi (FIG.3A) or Hs445 (FIG. 3B) cells in 100 μL normal saline were injectedintravenously (IV) into SCID mice. Mice were treated either with 200 ugintravenous rituximab twice weekly for 8 doses (coarse broken line - - -), 400 ug (1→3),(1→4)-D-β-glucan administered orally via intragastricgavage daily for 29 days (fine broken line . . . ) or a combination ofrituximab and (1→3),(1→4)-D-β-glucan(thin solid line), or left untreated(thick solid line) commencing 10 days after tumor implantation. Tumorsgrew systemically and mice became paralyzed when tumor cells infiltratedthe spinal canal, resulting in hind-leg paralysis. Mice were sacrificedat onset of paralysis or when animals lost 10% of their body weight.Kaplan-Maier survival curves for the various groups are shown in FIGS.2A (Daudi) and 2B (Hs445). Mice treated with a combination of (1→3),(1→4)-D-β-glucan and rituximab had a significantly increased survivalwhen compared to all other treatment groups (p<0.0005 for Daudi andp=0.001 for Hs445) or when compared to rituximab alone (p<0.0005 forDaudi and p=0.01 for Hs445). Median survival for mice with no treatment,rituximab alone, BG, and rituximab+BG groups was 27,71,43 and 124 daysrespectively for Daudi xenografts, and 12, 16, 31 and 243 daysrespectively for Hs445 xenografts.

FIG. 4. Dose response of 3G6 (anti-GD2 IgM antibody) in the presence ofbarley β-glucan in the treatment of human neuroblastoma. Two millionLAN1 neuroblastoma cells were xenografted subcutaneously in athymicBalb/c mice. Treatment started in groups of 5 mice each, 2 weeks aftertumor implantation when visible tumors reached 0.7-0.8 cm diameter. 3G6group (solid squares) was treated with 200 ug of intravenous 3G6injected through the retroorbital plexus twice weekly (M and Th). 3G6+BGgroup was treated with 200 ug i.v. 3G6 twice weekly plus oralbeta-glucan (BG) 400 ug daily by gavage for a total of 14-18 days. 3G6was administered in 3 different doses (open triangle 8 ug per dose, opensquare 40 ug, open circle 200 ug). BG group (solid circles) received 400ug oral beta-glucan alone. Tumor size was measured from the first day oftreatment, and the product of the largest diameters expressed as percentof the size on day 0 of treatment. Vertical bars represent standarderrors, depicted in only 4 groups for clarity. While BG alone and 3G6alone showed no anti-tumor effect, the BG+200 ug 3G6 group showed highlysignificant tumor shrinkage and suppression which was 3G6 dose-dependent(p<0.05).

FIG. 5. Treatment of human neuroblastoma using 3G6 (anti-GD2 IgMantibody) in the presence of yeast (1→3),(1→6)-β-D-glucan. Two millionLAN1 neuroblastoma cells were xenografted subcutaneously in athymicBalb/c mice. Treatment started in groups of 5 mice each, 2 weeks aftertumor implantation when visible tumors reached 0.7-0.8 cm diameter. 3G6group (solid squares) was treated with 200 ug of intravenous 3G6injected through the retroorbital plexus twice weekly (M and Th) for atotal of 5 doses. Particulate yeast glucan group (solid triangles)received 400 ug oral particulate yeast glucan alone. 3G6+whole yeastparticles (open diamond) was treated with 200 ug iv 3G6 twice weeklyplus yeast particles 400 ug daily by gavage for a total of 14-18 days.3G6+soluble yeast glucan group was treated with 200 ug iv 3G6 twiceweekly plus soluble yeast glucan 400 ug daily by gavage for a total of14-18 days. 3G6+particulate yeast glucan group was treated with 200 ugi.v. 3G6 twice weekly plus particulate yeast glucan 400 ug daily bygavage for a total of 14-18 days. Tumor size was measured from the firstday of treatment, and the product of the largest diameters expressed aspercent of the size on day 0 of treatment. Vertical bars representstandard errors, depicted in only 4 groups for clarity. While glucanalone and 3G6 alone showed no anti-tumor effect, soluble and particulateyeast glucan when combined with 3G6 group showed highly significanttumor shrinkage and suppression (p<0.05).

FIG. 6. Treatment of human neuroblastoma using 3F8 (anti-GD2 IgGantibody) in the presence of barley and yeast β-glucan. Two million LAN1neuroblastoma cells were xenografted subcutaneously in athymic Balb/cmice. Treatment started in groups of 5 mice each, 2 weeks after tumorimplantation when visible tumors reached 0.7-0.8 cm diameter. 3F8 group(solid diamonds) was treated with 200 ug of intravenous 3F8 injectedthrough the retroorbital plexus twice weekly (M and Th) for a total of 5doses. Barley glucan group (solid squares) received 400 ug barely glucanalone. 3F8+barley glucan group (open diamond) was treated with 200 ugi.v. 3F8 twice weekly plus barely glucan 400 ug daily by gavage for atotal of 14-18 days. 3F8+soluble yeast glucan group (open squares) wastreated with 200 ug iv 3F8 twice weekly plus soluble yeast glucan 400 ugdaily by gavage for a total of 14-18 days. Tumor size was measured fromthe first day of treatment, and the product of the largest diametersexpressed as percent of the size on day 0 of treatment. Vertical barsrepresent standard errors. While glucan alone and 3F8 alone showed noanti-tumor effect, barley and soluble yeast glucan when combined with3F8 group showed highly significant tumor shrinkage and suppression(p<0.05).

FIG. 7. Treatment of disseminating human lymphoma in SCID mice usingRituxan and barley or yeast β-glucan. 5×10e6 Daudi cells in 100 μlnormal saline were injected intravenously (IV) into SCID mice. Tumorsgrew systemically and mice became paralyzed when tumor cells infiltratedthe spinal canal, resulting in hind-leg paralysis. Mice were sacrificedat onset of paralysis or when animals lost 10% of their body weight.Therapy was initiated ten days after injection of tumor cells. 40 μgrituximab (Genentech, San Francisco, Calif.) was injected intravenouslytwice weekly for a total of eight injections and 400 μg glucanadministered orally via intragastric gavage daily for 29 days. Mice wereweighed weekly and observed clinically at least once daily. Micereceiving rituxan plus barley glucan or rituxan plus yeast solubleglucan have a highly significant prolonged survival (p<0.05).

FIG. 8. Illustrates the pEGP-C1 vector purchased from BD Biosciences(Palo Alto, Calif.).

FIG. 9. Shows glucan facilitates gene transfer into monocytes.

FIG. 10. Illustrates higher molecular weight β-glucan and gene transfer.

FIG. 11. Illustrates presence of GFP mRNA in circulating monocytes.

FIG. 12. Shows a ¹H NMR spectrum (cut-out from 2.7 to 5.5 ppm) of atypical SBG (Soluble Beta Glucan) (Biotec Pharamacon ASA, Tromsø,NORWAY) sample dissolved in DMSO-d₆ at a concentration of approximately20 mg/ml and with a few drops of TFA-d added. The spectrum was collectedover 2 hours on a JEOL ECX 400 NMR spectrometer at 80° C. Chemicalshifts were referenced to the residual proton resonance from the DMSO-d₆at 2.5 ppm, and the spectrum was baseline corrected.

FIG. 13. Shows a viscosity profile of a 2% solution of SBG at 20 or 30°C. at different shear rates. Glycerol (87%) is used as referencesolution.

DETAILED DESCRIPTION OF THE INVENTION

The following terms shall be used to describe the present invention. Inthe absence of a specific definition set forth herein, the terms used todescribe the present invention shall be given their common meaning asunderstood by those of ordinary skill in the art.

In the present invention the meaning of the expression “higher orderconformation” defines the formation of a molecule by transformation of acollection of free atoms, which find themselves in a random spatialconfiguration, into a more stable non-random pattern of those sameatoms. In other words these atoms have connected themselves to eachother, resulting in a new molecule, a new totality. The connection inthis case is chemical, i.e. by means of chemical bonds like hydrogenbonds. Several of such molecules can in turn react with each other,resulting in other molecules, either of the same size, or possibly of alarger size. Both are new totalities again. The larger molecules arehigher-order totalities.

In the present invention the expression “immunostimulating” describesthe effect of substances which stimulate the immune system by inducingactivation or increasing activity of any of its components.

In the present invention the expression “immunopotentiating” describesthe effect of substances which enhance or increase the effect of othersubstances used to stimulate the immune system.

The ability of beta-glucans to have immunopotentiating activity islikely the result of its ability to present multiple epitopes forinteraction with receptors on the target cells, thereby clusteringbeta-glucan receptors, mimicking the challenge by a pathogenic organism.Such multiple interactions with the specific receptors on the cell arebelieved to depend partly on glucan's ability to form “higher order”structures presenting multiple binding epitopes in close vicinity.Soluble beta-glucan formulations which possess durable interchainassociations, as expressed by a high viscosity profile, would thus belikely candidates for expressing “immunpotentiating” abilities.

The term “cancer” is used throughout the specification to refer to thepathological process that results in the formation and growth of acancerous or malignant neoplasm.

The term “effective amount” is used throughout the specification todescribe that amount of the compound according to the present inventionwhich is administered to an animal, especially a human, suffering fromcancer, to suppress or eradicate the growth or spread of the cancer.

The term “animal” is used throughout the specification to describe ananimal, preferably a mammal, more preferably a human, to whom treatmentor method according to the present invention is provided. For treatmentof those conditions or disease states which are specific for a specificanimal such as a human patient, the term patient refers to that specificanimal, and then the animal is specifically defined in the descriptionof this invention.

As used herein, the term “pharmaceutically acceptable carrier, additiveor excipient” means a chemical composition with which an appropriateglucan or derivative may be combined and which, following thecombination, can be used to administer the appropriate glucan to treatanimals, preferable mammals and most preferably humans.

Yeast-Derived Soluble Glucan Administered by Oral Route Enhances theEfficacy of Antibodies

Soluble glucan with the molecular structure where (1→3)-β-D-glucan unitsform the backbone with branches made up of (1→3)-β-D-glucan unitspositioned at (1→6)-β-D-glucan hinges was isolated from Baker's yeast,Saccharomyces cerevisiae. Mixed molecular weight fractions were obtainedand tested for synergy with monoclonal antibodies in tumor models. Theanti-tumor effect of soluble yeast beta-glucan was found to be as goodas the anti-tumor effect of soluble barley beta-glucan, when combinedwith monoclonal antibodies specific for human cancer as detailed below

Previously, i.e., in U.S. Ser. No. 60/261,911, it was shown that oraladministrated beta-1,3 and 1,4-glucans with high molecular weight andhigh viscosity profile isolated from barley is effective in enhancingthe efficacy of i.v. administered antibodies in eradication orsuppression of cancer or tumor cells, whereas the tested types ofbeta-1,3/1,6-linked glucans are less potent. The present invention nowdemonstrates that a composition of soluble beta-glucans withbeta-1,3-linkages having specific types of side chains and higher orderconformation giving a high viscosity profile than those used previously,surprisingly are equally active as barley derived beta-glucans.

The antibody used can be a single monoclonal antibody or a combinationof antibodies. The antibodies may be directed to at least one epitope ormultiple epitopes of an antigen or multiple antigens. Accordingly, thisinvention encompasses at least one antibody.

It is generally accepted that beta-glucans of microbial origin, likeyeasts, is recognised by specific pattern recognition receptors onimmune cells as a result from a phylogenetic adaptation for detectingpossible pathogens. Beta-glucans in e.g. fungal cell walls are the majorstructural element that secure the strength and integrity of the celland are thus vital for the organism. Beta-1,3-glucans are thus bothpresent in almost all fungal cells at the same time as they are highlyconserved structures, the latter being a prerequisite for so-calledPathogen Associated Molecular Patterns (PAMPs) recognised by the immunesystem. Immunologically active beta-glucans are likely to bind to thebeta-glucan receptor known as Dectin-1 when introduced to the organismthrough the gastrointestinal tract.

Purified beta-1,3-glucans having the structural elements andconformations mimicking its fungal origin as being recognised by theimmune cells would thus be considered to be favourable with respect toachieving an immune activation, especially when administered orally.Beta-1,3-glucans where these features have not been selected on wouldsubsequently be less active as also shown previously. It is likely thatbeta-1,3 and 1,4 glucans although not derived from a microbial organismwould interact with the immune cells based on its similarity toconserved structures on pathogenic organisms. As an illustrative exampleof a product asserting a pathogenic effect is particulate and solubleyeast cell wall glucans as described in PCT/IB95/00265 and EP 0759089.Other beta-1,3-glucan compositions having the ability to form interchainassociations, as exemplified in having a high viscosity profile asdescribed for the preferable barley beta-1,3-1,4-glucan preparations,would also be relevant candidates. Specific preparations of e.g.lentinan, scleroglucan and schizophyllan showing durable interchaininteractions are likely to be effective. Likewise would beta-1,3-glucanformulations solublised by deriatization, like glucan phosphates, glucansulphates, carboxymethyl-glucans, and retaining the immunopotentialtingactivity of the native molecule and interchain assosiation be possibleactive products.

Beta-glucan formulations not presenting a pathogen like feature, couldhowever be a potent adjuvant for immunotherapy when administereddirectly into systemic distribution, like when given i.v. as describedin Herlyn, D., Kaneko, Y., Powe, J., Aoki, T., & Koprowski, H. (1985)Monoclonal antibody-dependent murine macrophage-mediated cytotoxicityagainst human tumors is stimulated by lentinan. Jpn. J. Cancer Res., 76,37-42, or when given i.p. as described in U.S. Ser. No. 60/261,911.

In the present application it is disclosed a composition for achieving asynergistic therapeutic effect in an animal, preferably a mammal, mostpreferably a human in need thereof, comprising a viscous andimmunopotentiating beta-glucan composition comprising a beta-1,3-linkedbackbone as described in the general formula in Figure A and anantitumor antibody administrated to an animal, preferably a mammal, mostpreferably a human where the synergistic therapeutic effect is theeradication or suppression of cancer or tumor cells. The ability ofbeta-glucans to have immunopotentiating activity is likely the result ofits ability to present multiple epitopes for interaction with receptorson the target cells, thereby clustering beta-glucan receptors, mimickingthe challenge by a pathogenic organism. Such multiple interactions withthe specific receptors on the cell are believed to depend partly onglucan's ability to form “higher order” structures presenting multiplebinding epitopes in close vicinity. Soluble beta-glucan formulationswhich possess durable interchain associations, as expressed by a highviscosity profile, would thus be likely candidates for expressing“immunpotentiating” abilities.

A composition comprising beta-glucans where the beta-1,3-linked mainchain has a molecular weight (MW)>6000 Da, and has side chains attachedthereto giving a soluble product with strong interchain assosiations, ispreferable. Beta-1,3-glucans with beta-1,3-linked side chains anchoredto the main chain through a single beta-1,6-linkage that can be isolatedfrom yeast species like Bakers yeast, as the example shown in Figure Bwould be preferred. In contrast, beta-1,3-glucans from yeast havingrepetitive beta-1,6-linkages in the side chains as described byOnderdonk et al (Infection and Immunity, 1992, 60:1642-47) calledPoly-betal-6-glucotriosyl-betal-3-glucopyranose glucan (PGG orBetafectin) would be less active in this respect in light of the papersof Bohn and Bemiller (See Bohn, J. A. & BeMiller, J. N. (1995)(1-3)-b-D-glucans as biological response modifiers: a review ofstructure-function relationships. Carbohydrate Polymers, 28, 3-14.) andEngstad (See Engstad, R. E. & Robertsen, B. (1995) Effect ofstructurally different beta-glucans on immune responses in Atlanticsalmon (Salmo salar L.). Journal of Marine Biotechnology, 3, 203-207).Similarly, beta-1,3-glucan preparations forming isolated triple helicalconformations with weak interhelical associations lack the ability toform higher order conformations, and may be less active since they areunable to present a pathogen like expression (see Zimmerman et al.(1988) A novel-carbohydrate-glycosphingolipid interaction between abeta-(1-3)-glucan immunomodulator, PGG-glucan, and lactosylceramide ofhuman leukocytes: J Biol Chem 273:22014-22020).

An example of a highly active composition of beta-glucans in combinationwith antitumor antibodies is a mixture of soluble beta-glucan chainswith MW>6000 wherein the chains interact giving a higher orderconformation that would facilitate the immunostimulatory activity neededwhen administered orally for inducing a synergistic effect with theantibodies, wherein said mixture of soluble beta-glucans comprise linearbeta-1,3-glucan chains with a MW>6000 Da, or preferably, with MW rangingfrom 6000-15,000 Da, together with branched high molecular weightbeta-1,3-glucan (MW>15,000 Da) with beta-1,3 linked side chain(s) asdescribed in Figure B wherein the branches extend from within the mainchain. An example of the glucan as described above is SBG (Soluble BetaGlucan) produced by Biotec Pharamacon ASA (Tromsø, NORWAY). SBG isolatedfrom Bakers yeast and described by the NMR-spectra in FIG. 12 was shownto be as least as efficient than beta-1,3 and 1,4-linked glucan derivedfrom barley having the desired high viscosity profile. FIG. 12 shows acomplex beta-glucan composition with high molecular weight chains havingbeta-1,3-linked side chains attached to the repeating beta-1,3-linkedmain chain through a beta-1,6-linked branching point, and mediummolecular weight linear beta-1,3-glucan chains in the range of 6-15 kDa.SBG presents durable interchain assosiaction as demonstrated by its highviscosity profile and gelling behavior (see FIG. 13). SBG has been shownto be a potent immunostimulating agent for activating human leukocytesin vitro (see Engstad, C. S., Engstad, R. E., Olsen, J. O., & Osterud,B. (2002) The effect of soluble beta-1,3-glucan and lipopolysaccharideon cytokine production and coagulation activation in whole blood. Int.Immunopharmacol., 2, 1585-1597.), and also for modulating immunefunctions when given p.o. (see Breivik, T., Opstad, P. K., Engstad, R.,Gundersen, G., Gjermo, P., & Preus, H. (2005) Solublebeta-1,3/1,6-glucan from yeast inhibits experimental periodontal diseasein Wistar rats. J. Clinical Periodontology, 32, 347-3.).

Other structures and/or structural conformations in the composition ofbeta-1,3-glucans as described above can be readily identified orisolated by a person of ordinary skill in the art following the teachingof this invention, and is expected to have similar therapeutic effectwhen administered throughdifferent routes other than p.o. The above isthus a guideline to achieve a highly potent product, but is not alimitation towards even more potent products. Isolated structuralelements of the complex mixture as described above are expected to haveimproved effects over the present formulation when administered orally.

Products having the desired structural features giving a higher orderconformation like SBG that facilitates the needed interaction withresponding cells in the intestinal tract would be the preferred productswhen administered orally. Their action as immunopotentiators in synergywith anti-cancer antibodies is likely to be at least as powerful whenadministered parenterally, e.g. when administered i.p., s.c., i.m. ori.v. When administered orally the functional dose range would be in thearea of 1-500 mg/kg b.w. (body weight)/day, more preferable 10-200 mg/kgb.w./day, and most preferable 20-80 mg/kg/day. When administeredparenterally the functional dose range would be 0.1-10 mg/kg b.w./day.

Typically, dosages of the compound of the invention which may beadministered to an animal, preferably a human, will vary depending uponany number of factors, including but not limited to, the type of animaland type of cancer and disease state being treated, the age of theanimal, the route of administration and the relative therapeutic index.

The route(s) of administration will be readily apparent to the skilledartisan and will depend upon any number of factors including the typeand severity of the disease being treated, the type and age of theveterinary or human patient being treated, and the like.

Formulations suitable for oral administration of the beta-glucaninclude, but are not limited to, an aqueous or oily suspension, anaqueous or oily solution, or an emulsion. Such formulations can beadministered by any means including, but not limited to, soft gelatincapsules.

Liquid formulations of a pharmaceutical composition of the inventionwhich are suitable for oral administration may be prepared, packaged,and sold either in liquid form or in the form of a dry product intendedfor reconstitution with water or another suitable vehicle prior to use.

In general, the beta-glucan can be administered to an animal asfrequently as several times daily, or it may be administered lessfrequently, such as once a day. The antibody treatment will for instancedepend upon the type of antibody, the type cancer, the severity of thecancer, and the condition of each patient. The beta-glucan treatment isclosely interrelated with the antibody treatment regimen, and could beahead of, concurrent with, and after the antibody administration. Thefrequency of the beta-glucan and antibody dose will be readily apparentto the skilled artisan and will depend upon any number of factors, suchas, but not limited to, the type and severity of the disease beingtreated, the type and age of the humans. The treatment with thesubstance of the present invention could happen at the same time or atdifferent times. As an example, the beta-glucan treatment could alsostart a few days ahead of the i.v. antibody treatment, and then thebeta-glucan is administered concurrently with the antibody. In anembodiment, beta-glucan treatment continues for a few days after endingthe antibody treatment. The antibody treatment could include a cocktailof antibodies or antibody-formulations, and/or modified antibodiesand/or derivatives thereof.

When administered orally, glucan is taken up by macrophages andmonocytes which carry these carbohydrates to the marrow andreticuloendothelial system from where they are released, in anappropriately processed form, onto myeloid cells including neutrophils,and onto lymphoid cells including natural killer (NK) cells. Thisprocessed glucan binds to CR3 on these neutrophils and NK cells,activating them in tumor cytotoxicity in the presence of tumor-specificantibodies.

Since macrophage and monocytes ingest glucan (whether soluble, gel orparticle) from the gut, glucan is a potential conduit for gene therapy.Unlike proteins, DNA or plasmids are relatively heat-stable, and can beeasily incorporated into warm soluble barley glucan which gels whencooled to room or body temperature.

It has been shown that when mice are fed these DNA-glucan complexes,reporter genes can be detected in peripheral blood monocytes andmacrophages within days. More importantly these reporter genes areexpressed in these cells, a few days after ingestion of these DNAcomplexes.

This invention provides a conduit for delivering DNA or plasmids intothe human body. In an embodiment, the conduit is glucan or similarcarbohydrates capable of interacting with and protecting the DNA orplasmids for efficient uptake of the said DNA or plasmids into therelevant immune cells. Soluble or orally administered glucan can be usedas a convenient vehicle for correcting genetic defects of the relevantimmune cells, or for administering genetic vaccines.

As it can easily be appreciated by an ordinary skilled artisan, othercarbohydrates capable of functioning like glucan could be identified andused in a similar fashion. One easy screening for such carbohydrates canbe established using glucan as the positive control.

The glucan includes but is not limited to β(1-3) and β(1-4) mixedlinkage-glucan. In an embodiment, the glucan has a high molecularweight. In another embodiment, the glucan has β(1-3) and β(1-6) linkagesand are able to form complex conformations interacting with thesubstance to be delivered.

This invention also provides a method for introducing substance intocells comprising contacting glucans comprising the substance to bedelivered with said cells. One can use reporter genes or other markersto assess the efficiency of the said introduction. Reporter genes ormarkers are well known in the molecular biology field. In addition, thisinvention provides a method for introducing substance into a subjectcomprising administering to the subject an effective amount of a glucancomprising the substance to be delivered.

This invention provides a composition for the oral delivery of one ormore substances comprising an effective amount of an orally administeredbeta-glucan and one or more chemotherapeutic agents.

In an embodiment, the glucan contains 1,3-1,6 or 1,3-1,4 mixed linkages,or a mixture of 1,3-1,6 and 1,3-1,4 mixed linkages. In anotherembodiment, the glucan enhances the efficacy of chemotherapeutic agentsor anti-cancer antibodies.

In a further embodiment, the glucan is derived from grass, plants,mushroom, yeast, barley, fungi, wheat or seaweed. In a furtherembodiment, the glucan has high molecular weight. In a furtherembodiment, the molecular weight of the glucan is at least 6,000Daltons.

In a further embodiment, the substance which can be delivered by glucanis a peptide, protein, RNA, DNA, plasmid, or chemotherapeutic agent. Asused herein, chemotherapeutic agents include chemicals that combatdisease in the body of an animal, preferably a mammal, most preferably ahuman or medications used to treat various forms of cancer.

This invention provides a method for treating a subject with geneticdisorder comprising administering to the subject an effective amount ofthe above-described glucan and a substance capable of correcting saidgenetic disorder, wherein the substance is incorporated into the glucanto allow delivery of the substance by oral route. The substance includesbut is not limited to a peptide, protein, RNA, DNA, plasmid and othersmall molecule and compound.

This invention provides a composition comprising an effective amount oforally administered (1→3),(1→6) beta-glucan capable of enhancingefficacy of antibodies. Glucans derived from cell walls of yeasts, suchas Saccharomyces cervisiae, may be used in the above-describedcompositions. Preferably, Glucans having β(1-3) and β(1-6) linkages suchas SBG is used in the above-described compositions.

The above mentioned pharmaceutical compositions may containpharmaceutically acceptable carriers and other ingredients known toenhance and facilitate drug administration.

Such a pharmaceutical composition may comprise the active ingredientalone, in a form suitable for administration to a subject, or thepharmaceutical composition may comprise the active ingredient and one ormore pharmaceutically acceptable carriers, one or more additionalingredients, or some combination of these. The active ingredient may bepresent in the pharmaceutical composition in forms which are generallywell known in the art.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a carrier or one ormore other accessory ingredients, and then, if necessary or desirable,shaping or packaging the product into a desired single- or multi-doseunit.

The relative amounts of the active ingredient, the pharmaceuticallyacceptable carrier, and any additional ingredients in a pharmaceuticalcomposition of the invention will vary, depending upon the identity,size, and condition of the subject treated.

In addition to the active ingredient, a pharmaceutical composition ofthe invention may further comprise one or more additionalpharmaceutically active agents, including other anti-cancer agents.Controlled- or sustained-release formulations of a pharmaceuticalcomposition of the invention may be made using conventional technology.

In an embodiment, the antibody is a monoclonal antibody, or an antibodyagainst cancer or tumor cells, which include but are not limited toanti-CEA antibody, anti-CD20 antibodies, anti-CD25 antibodies, anti-CD22antibodies, anti-HER2 antibodies, anti-tenascin antibodies, MoAb M195,Dacluzimab, anti-TAG-72 antibodies, R24, Herceptin, Rituximab, 528, IgG,IgM, IgA, C225, Epratuzumab, and MoAb 3F8. In another embodiment, theantibody is a tumor-binding antibody.

In another embodiment, the antibody is capable of activating complementand/or activating the antibody dependent cell-mediated cytotoxicity. Ina further embodiment, the antibody modulates T-cell or B-cell function.

In a further embodiment, the antibody is directed at the epidermalgrowth factor receptor, a ganglioside, such as GD3 or GD2.

In a further embodiment, the antibodies are effective against cancerswhich include neuroblastoma, melanoma, non-Hodgkin's lymphoma,Epstein-Barr related lymphoma, Hodgkin's lymphoma, retinoblastoma, smallcell lung cancer, brain tumors, leukemia, epidermoid carcinoma, prostatecancer, renal cell carcinoma, transitional cell carcinoma, breastcancer, ovarian cancer, lung cancer, colon cancer, liver cancer, stomachcancer, or other gastrointestinal cancers.

In a further embodiment, the above-described composition is in apharmaceutically acceptable carrier.

This invention provides a method for treating a subject comprisingadministrating the above-described composition to a subject.

This invention provides a composition comprising an effective amount oforally administered (1→3),(1→6) beta-glucan capable of enhancingefficacy of vaccines. In an embodiment, the vaccine is against cancer orinfectious agents, such as bacteria, viruses, fungi, or parasites.

This invention provides a composition comprising an effective amount ofbrally administered (1→3),(1→6) beta-glucan capable of enhancingefficacy of natural antibodies or infectious agents.

This invention provides a composition comprising an effective amount oforally administered (1→3),(1→6) beta-glucan capable of enhancing hostimmunity.

This invention provides a composition comprising an effective amount oforally administered (1→3),(1→6) beta-glucan capable of enhancing theaction of an agent in preventing tissue rejection. In an embodiment, thetissue is transplanted tissue or transplanted organ or the host as ingraft-versus-host disease.

In an embodiment, the glucan of the above-described composition has highmolecular weight. The molecular weight of glucan is at least 10,000Daltons. In another embodiment, the glucan is derived from barley, oat,mushroom, seaweed, fungi, yeast, wheat or moss. In a further embodiment,the glucan is stable to heat treatment.

In a further embodiment, above-describe composition is stable afterboiling for 3 hours. In an embodiment, the effective dose of theabove-described composition is about >=25 mg/kg/day, five days a weekfor a total of 2-4 weeks.

This invention also provides kits for Inhibiting Cancer Cell Growthand/or Metastasis. The invention includes a kit or an administrationdevice comprising a glucan as identified in the invention and aninformation material which describes administering the glucan or acomposition comprising the glucan to a human. The kit or administrationdevice may have a compartment containing the glucan or the compositionof the present invention. As used herein, the “Information material”includes, but is not limited to, for instance a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of the composition of the inventionfor its designated use.

The above described invention relates to the administration of anidentified compound in a pharmaceutical composition to practice themethods of the invention, the composition comprising the compound or anappropriate derivative or fragment of the compound and apharmaceutically acceptable carrier, additive or excipient.

The invention will be better understood by reference to the ExperimentalDetails which follow, but those skilled in the art will readilyappreciate that the specific experiments detailed are only illustrative,and are not meant to limit the invention as described herein, which isdefined by the claims which follow thereafter.

Exemplification

The invention being generally described, will be more readily understoodby reference to the following examples which are included merely forpurposes of illustration of certain aspects and embodiments of thepresent invention, and are not intended to limit the invention.

Phase I Study of Barley β-Glucan in Combination with Anti-GD2 Antibodyin Stage 4 Neuroblastoma

A total of 24 patients were studied. These patients are all children oradolescents with relapsed or refractory stage 4neuroblastoma metastaticto bone, marrow or distant lymph nodes, some with large soft tissuemasses. Beta-glucan was well tolerated with no dose-limiting toxicities.Anti-tumor responses were recorded for marrow disease (histology, MIBGscans), soft tissue tumors (CT), as well biochemical markers (urine VMAand HVA tumor markers). One example of tumor response is shown in FIGS.1A and 1B: ¹³¹I-metaiodobenzylguanidine (MIBG) scans showingnear-complete resolution of extensive metastases after one treatmentcycle of 3F8 plus beta-glucan. These responses are uncommon in patientswith refractory or relapsed metastatic stage 4 NB treated with 3F8 aloneor 3F8 in combination with cytokines. The best response rate for 3F8 todate was in a Phase II trial of combination 3F8 plus GMCSF where 7 of 33(21%) children achieved MIBG improvement. In contrast, 62% (13 of 21)evaluable patients on 3F8+beta-glucan had MIBG improvement, a neartripling of the response rate (p=0.008 by χ²). In addition, among 15patients with marrow disease, 5 achieved complete marrow remission(30%), and 8 with stable disease in the marrow. (See FIG. 1)

Synergism Between Soluble Beta-Glucan and Rituximab Against Lymphoma

Rituximab activates complement-mediated and antibody-dependentcell-mediated cytotoxicities, and is effective against B-cell lymphomas.Beta-glucans are naturally occurring glucose polymers that bind to thelectin domain of CR3, a receptor widely expressed among leukocytes,priming it for binding to iC3b activated by antibodies. Barley-derived(1→3),(1→4)-β-D-glucan (BG), when administered orally (400 μg per day×29days), strongly synergized with subtherapeutic doses of intravenousrituximab (200 μg twice/week×8 doses) in the therapy of CD20-positivehuman lymphomas. Growth of established subcutaneous non-Hodgkin'slymphoma (NHL) (Daudi and EBV-derived B-NHL) or Hodgkin's disease (Hs445or RPMI6666) xenografted in SCID mice was significantly suppressed, whencompared to mice treated with rituximab or BG alone. Survival of micewith disseminated lymphoma (Daudi and Hs445) was significantlyincreased. There was no weight loss or clinical toxicity in treatedanimals. The results demonstrate that the therapeutic efficacy and lackof toxicity of BG plus rituximab.

Study Design

Cell Lines:

Human Burkitt's lymphoma cell line, Daudi, and Hodgkin's disease (HD)cell lines Hs445 and RPMI 6666 were purchased from American Type CultureCollection (Rockville, Md.). Human EBV-BLCL were established usingpreviously described methods (³⁷).

Mice:

Fox Chase ICR SCID mice (Taconic, White Plains, N.Y.) were maintainedunder institutionally approved guidelines and protocols.

Tumor Models:

Subcutaneous tumors were established by injecting 5×10⁶ cells suspendedin 0.1 ml of Matrigel (Becton-Dickinson, Franklin Lakes, N.J.) into miceflanks. Tumor dimensions were measured two to three times a week andtumor size was calculated as the product of the two largest diameters.Mice were sacrificed when maximum tumor dimension exceeded 20 mm. Adisseminated tumor model was established in SCID mice as previouslydescribed (³⁸). Briefly, 5×10⁶ Daudi or Hs445 cells in 100 μl normalsaline were injected intravenously into SCID mice. Tumors grewsystemically and mice became paralyzed when tumor cells infiltrated thespinal cord, resulting in hind-leg paralysis. Mice were sacrificed atonset of paralysis or when animals lost 10% of their body weight.

Treatment Regimens:

For mice with subcutaneous tumors, therapy was initiated after tumorswere established (7-8 mm diameter). For the disseminated tumor model,therapy was initiated ten days after injection of tumor cells. Groups ofat least five mice per treatment regimen received either rituximab, BG,neither or both. 200 μg rituximab (Genentech, San Francisco, Calif.) wasinjected intravenously twice weekly for a total of eight injections and400 μg BG (Sigma, St. Louis, Mo.) administered orally via intragastricgavage daily for 29 days. Animals were weighed weekly and observedclinically at least once daily.

Statistical Analysis:

Tumor growth was calculated by fitting a regression slope for eachindividual mouse to log transformed values of tumor size. Slopes werecompared between groups using t-tests using a previously describedmethod for censored observations (³⁹).

Survival in mice with disseminated disease was compared usingKaplan-Meier analysis and proportion of deaths was compared by Fisher'sexact χ2 test. Analyses were conducted using STATA 7 (Stata Corporation,College Station, Tex.).

Results and Discussion

In all subcutaneous xenograft models, significant reduction in tumorgrowth was noted in mice treated with a combination of rituximab and BG.Mice treated with rituximab alone showed a modest reduction in tumorgrowth, while those treated with BG alone or left untreated had unabatedtumor growth (FIG. 1A, 1B, 1C). All tumors except for those treated withcombination therapy grew beyond 20 mm size and mice had to besacrificed. Mice on combination treatment had persistent tumorsuppression even after treatment was stopped. In a multivariable linearmodel of tumor growth rate, using dummy variables for treatment, theinteraction between BG and rituximab was positive and significant,demonstrating synergism.

For disseminated xenografts, there was a significant difference insurvival between the combination and control groups for both NHL and HDmodels (p<0.005, by log-rank) (FIG. 2). 5/38 mice and 2/8 mice withdisseminated Daudi and Hs445 tumors respectively treated withcombination BG and rituximab were surviving >12 months after therapy wasdiscontinued suggesting complete eradication of disease. In contrast,0/29 and 0/8 mice receiving rituximab alone in respective groupssurvived (15% vs. 0% survival; χ2=0.01). There was no significant weightloss or other clinically apparent adverse effects. That BG is absorbedcan be inferred from the fact that it could be detected intracellularlywithin fixed and permeabilized peripheral blood leucocytes byimmunofluorescence.

In these studies, synergism between BG and rituximab was highlysignificant irrespective of the type of CD20-positive lymphoma. Improvedresponses in Daudi xenografts as compared to Hs445 may be attributableto higher CD20 expression in the former (Mean geometric fluorescencechannel for Daudi 241 compared to 184 for Hs445). When tumors thatprogressed were examined for CD20 expression by immunofluorescencestudies of single cell suspensions or indirect immunohistochemistry offrozen sections, no significant difference was noted between groupstreated with rituximab, BG alone or rituximab+BG, indicating thattreatment with rituximab+BG was not associated with loss of CD20.

Synergism between other complement-activating monoclonal antibodies andBG (^(35,36)) were previously demonstrated. The current data extend thisobservation to rituximab. CDC is considered an important mechanism forrituximab cytotoxicity. Rodent complement is not inhibited efficientlyby human complement regulatory proteins (mCRP). Therefore CDC can be aneffective anti-tumor mechanism in xenograft models. However in a study,at sub-therapeutic doses of antibody, rituximab-mediated ADCC and CDCwere not sufficient to effect tumor cell killing. Since BG has no directeffect on ADCC (⁴⁰), this synergy is most likely a result ofiC3b-mediated tumor cytotoxicity. Lymphoma cells express mCRP includingCD46, CD55, and CD59 (^(30,41)) However, iC3b-mediated cytotoxicity isunaffected by the presence of CD59 which affects only MAC-mediatedcomplement cytotoxicity (⁴²). Furthermore, in human breast carcinomatumors, deposition of iC3b has been demonstrated despite the presence ofmCRP ⁽⁴³) indicating that unlike their inhibitory effect on MAC, effecton iC3b-mediated tumor cytotoxicity is not absolute.

If this synergistic effect can be safely reproduced in humans,iC3b-mediated cytotoxicity may be a potential strategy to overcomerituximab resistance in patients with B-cell malignancies. Since neitherT nor B cells are required for this synergistic effect, BG may have apotential role even in immunocompromised lymphoma patients. Furthermore,in patients with autoimmune disorders, B-cell depletion may be enhancedwith this non-toxic oral therapy. Conversely, beta-glucans can enhancerelease of cytokines such as TNF-α and IL-6 (⁴⁴), and because the acutetoxicities of rituximab are also related to cytokine release secondaryto complement activation (⁴⁵), there is a potential of increasedtoxicity when BG and rituximab are used in combination. Carefullydesigned phase I studies are necessary in order to define the safety andefficacy in developing BG as an adjunct to rituximab therapy in thetreatment of B-cell disorders and in antibody-based therapies of othercancers.

Oral β-Glucan Synergizes with IgM Antibodies

Natural IgM antibody from human serum when administered i.v. wascytotoxic for human neuroblastoma (NB) cells effecting growth arrest ofsubcutaneous solid human NB xenografts in nude rats (^(46,47)). IgM wastaken up by the tumors with massive perivascular complement activationand accumulation of granulocytes after 24 hours (⁴⁸). In metastatic NBmodel, IgM antibody was effective in eliminating tumors in 90% of themice (⁴⁹). The absence of this anti-NB IgM antibody during infancy andamong NB patients (of any age), and its prevalence after 12 months ofage has raised the hypothesis that natural IgM antibodies could play arole as an immunological control mechanism against NB (⁵⁰). 3G6 is ananti-GD2 mouse IgM monoclonal antibody (MoAb). Within 48 hours afteri.v. injection of biotinylated 3G6, subcutaneous NB xenografts showedmembrane staining of tumor cells. Although 3G6 had lower meanfluorescence (53±19 fluorescent channel units, n=7 mice) when comparedto 3F8, an IgG MoAb (149±44, n=7), 3G6 plus beta-glucan was effectiveagainst sc human NB (p<0.05), with a dose response curve (FIG. 4)comparable to that of 3F8 (³⁵). These findings were consistent withthose using human natural anti-NB IgM (^(46,47)). These datademonstrates that oral beta-glucan enhances not just IgG inducingvaccines, but also IgM inducing vaccines.

Soluble (1→3),(1→6) B-Glucan is Effective in Enhancing Antibody Therapyof Cancer

LAN-1 tumor cells were planted (2×10⁶ cells) in 100 μl of Matrigel(Sigma) subcutaneously. Tumor dimensions were measured two to threetimes a week with vernier calipers, and tumor size was calculated as theproduct of the two largest perpendicular diameters. All treatmentstudies started in groups of 4-5 mice when tumor diameters reached 0.7to 0.8 cm. Mice received antibody (3F8 or 3G6) treatment (200 ug perday) i.v. (by tail vein injection) twice weekly×5 doses and oralbeta-glucan (400 ug per day) by intragastric injection every day for atotal 14-18 days. See FIGS. 5 and 6.

In similar experiments a subcutaneous lymphoma model was studied. Here5×10⁶ cells suspended in 0.1 ml of Matrigel (Becton-Dickinson, FranklinLakes, N.J.) were planted into mice flanks. Tumor dimensions weremeasured two to three times a week and tumor size was calculated asproduct of the two largest diameters. Mice were sacrificed when maximumtumor dimension exceeded 20 mm. 200 μg rituximab (Genentech, SanFrancisco, Calif.) was injected intravenously twice weekly for a totalof eight injections and 400 μg glucan administered orally viaintragastric gavage daily for 29 days. Mice were weighed weekly andobserved clinically at least once daily.

These series of subcutaneous tumor models showed that soluble yeast(1→3),(1→6) beta-glucan is at least as potent as barley (1→3),(1→4)beta-glucan. In addition, the source and physical form of yeast glucancan make a substantial difference in its activity.

Metastatic lymphoma model was also studied. A model of disseminatedtumors was established in SCID mice as previously described (³⁸).Briefly, 5×10⁶ Daudi cells in 100 μl normal saline were injectedintravenously (i.v.) into SCID mice. Tumors grew systemically and micebecame paralyzed when tumor cells infiltrated the spinal canal,resulting in hind-leg paralysis. Mice were sacrificed at onset ofparalysis or when animals lost 10% of their body weight. Therapy wasinitiated ten days after injection of tumor cells. 40 μg rituximab(Genentech, San Francisco, Calif.) was injected intravenously twiceweekly for a total of eight injections and 400 μg glucan administeredorally via intragastric gavage daily for 29 days. Mice were weighedweekly and observed clinically at least once daily. See FIG. 7.

Again both barley glucan and yeast glucan showed significant effect whencombined with Rituxan. Neither barely glucan nor yeast glucan has anyeffect on survival when used alone.

Mechanism by Which Orally Administered β-Glucans Function withAnti-Tumor Monoclonal Antibodies to Mediate Tumor Regression (⁵¹)

Using syngeneic tumor (GD2+ RMA-S) in wild type (WT) C57B1/6 mice versuseither CR3-deficient (CD11b −/−) or C3-deficient (C3 −/−) C57B1/6 mice,MoAb alone elicited no tumor regression, whereas combining the i.v.anti-GD2 MoAb with oral barley or yeast beta-glucan elicited significantregression in WT but not in CR3-deficient mice. Moreover, the combinedtreatment with i.v. MoAb and oral beta-glucans produced 60-100%tumor-free survivors in WT mice, but only 0-20% survival in theCR3-deficient mice. These experiments demonstrated a near absoluterequirement for leukocyte CR3 for the anti-tumor effect, especially whenoral barley beta-glucan was given with anti-tumor MoAb. A therapyprotocol comparing WT to C3-deficient mice similarly showed that oralbeta-glucan therapy required serum C3. When barley beta-glucan and yeastbeta-glucan were labeled with fluorescein (BG-F and YG-F) and given tomice by intragastric injection, the trafficking of beta-glucan wasfollowed. Within three days of daily oral administration of BG-F orYG-F, macrophages in the spleen and lymph nodes containedfluorescein-labeled beta-glucan. After 4 d, YG-F and BG-F were alsoobserved in macrophages in bone marrow. When the uptake of YG-F and BG-Fby WT versus CR3-deficient mice was compared, no differences wereapparent in either the percentage of macrophages containing ingestedbeta-glucan-F or the amount of beta-glucan-F per cell. Thus, the uptakeof barley and yeast beta-glucan by gastrointestinal macrophages does notrequire CR3 and is-likely mediated instead by Dectin-1 (⁵²). Macrophagesin vitro and in the marrow were able to degrade large molecules ofbarley or yeast beta-glucan into smaller biologically-active fragmentsof beta-glucan that are then released.

To determine if the soluble beta-glucan-F released by macrophages hadindeed been taken up by bone marrow granulocytes, groups of WT orCR3-deficient mice that had been given YG-F or BG-F for 10 days wereinjected i.p. with thioglycolate medium to elicit the marginated pool ofbone marrow granulocytes into the peritoneal cavity. Only WTgranulocytes were able to pick up the YG-F and BG-F released frommacrophages. These data suggest a sequential ingestion of beta-glucan bygastrointestinal macrophages that shuttle the beta-glucan to the bonemarrow where soluble degradation fragments are released and taken up bygranulocytes via membrane CR3. When peritoneal granulocytes wereisolated from WT and CR3-deficient mice that had been given oralbeta-glucan, only WT granulocytes were able to kill iC3b-coated tumorcells in vitro. These experiments show that bone marrow granulocytes andtissue macrophages acquire membrane CR3-bound soluble beta-glucan fromgastrointestinal macrophages, and that this bound beta-glucan primes theCR3 of both granulocytes and macrophages so that when they are recruitedto a site of inflammation they are able to kill iC3b-coated tumor cells.

Soluble β-Glucan as a Conduit for Plasmids

The major obstacles for the delivery of DNA, RNA and proteins orally arethe acidic and proteolytic environment of the stomach, and limiteduptake of proteins by the GALT. It is believed that M cells within thePeyer's patches and phagocytes are the predominant vehicles for uptakeof microparticulates. However, nanoparticles may also access GALT via aparacellular mechanism (^(53,54)) and by transcytosis (35). In eithercase, particle uptake observed can be improved using particles withmucoadhesive properties or affinity for receptors on cells.

Many polymers have been used to fabricate nanoparticles aremucoadhesive. Among them are alginate, carrageenans, and pectin.Although these materials were often used as the core polymers innanoparticulates, no specific receptor has been identified for thesepolymers and the efficiency of uptake remains suboptimal. Dectin-1 isnow known to be a universal receptor for β-glucan, and is found in manyhuman tissues including monocytes and phagocytes. The gelling propertiesof high molecular weight β-glucan allows RNA, DNA and proteins to beembedded. Since sugars are highly resistant to acid conditions andenzymes, proteins, RNA and DNA remain protected during their passagethrough the gastrointestinal tract. Through the high affinity Dectin-1receptor for β-glucan, these substances can be introduced into thephagocytes as potential vehicles to the rest of the body.

The pEGP-C1 vector (See FIG. 8) was purchased from BD Biosciences (PaloAlto, Calif.) and prepared according to manufacturers' instructions.pEGFP-C1 encodes a red-shifted variant of wild-type GFP (1-3) which hasbeen optimized for brighter fluorescence and higher expression inmammalian cells. (Excitation maximum=488 nm; emission maximum=507 nm.)The vector backbone also contains an SV40 origin for replication inmammalian cells only if they express the SV40 T-antigen. A bacterialpromoter upstream of this cassette expresses kanamycin resistance in E.coli. The pEGFP-C1 backbone also provides a pUC origin of replicationfor propagation in E. coli and an f1 origin for single-stranded DNAproduction.

Mice were fed with 50 μg pEGFP-c1 plasmid mixed into 400 μg beta-glucan(˜200,000 Daltons) in 100 μl saline by oral gavage while control micewere given plasmid alone. Oral feeding was done for 3 consecutive days(days 1, 2 and 3). 50 μl blood taken from tail vein were analyzed byFCAS analysis after lysis of RBC and the % of GFP-expressing cells inthe monocyte population were recorded. The mean ratio of % green cellsin glucan versus no glucan groups (n=4-9 mice per group) is presented inFIG. 9. Throughout the 14 days of the experiment, % green monocytes inthe no-glucan group remained stable at background levels. On the otherhand, after day 1 of oral gavage, there was a consistent higher % ofcirculating green monocytes, which peaked around day 8. Since the GFP isnot normally found in mouse monocytes, the presence of green cells isconsistent with GFP protein expression following entry of the plasmidinto the monocytes which circulate in the blood.

The experiment was repeated using barley β-glucan of higher molecularweight (˜350,000 Daltons) with better gelling properties. In FIG. 10,similar kinetics was seen, with a higher percent of green cells thatpersisted from day 8 through day 11 (n=4 mice per group).

Presence of GFP mRNA was tested using quantitative reverse-transcriptionPCR analysis. Mice were fed with 50 μg pEGFP-c1 plasmid mixed into 400μg high molecular weight (˜350,000 Daltons) beta-glucan in 100 μl salineby oral gavage while control mice were given plasmid alone. 50 μlperipheral blood was used to extract total RNA, reverse transcribed andquantitative real-time PCR was performed using a modification of themethod previously described (⁵⁶). The house keeping gene mouse GAPDH isused as internal control. Transcript level is calculated using a knownGFP and GAPDH standard. Transcript units are calculated separately forGFP and GAPDH and results as a ratio of GFP over GAPDH. In FIG. 11, themean RNA level (GFP/GAPDH) is expressed as a ratio of glucan versus noglucan groups (n=4 mice per group). GFP mRNA was detected up to day 10.

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1-11. (canceled)
 12. A composition for delivering an isolated DNA, RNAor plasmid into cells, comprising said DNA, RNA or plasmid and aneffective amount of a yeast β-glucan, wherein said β-glucan is notencapsulated.
 13. The composition of claim 12, wherein the glucancomprises β-1,3 bonds in the backbone.
 14. The composition of claim 13,wherein the glucan further comprises branches of β-1,3-linked glucoseunits, attached to the backbone via β-1,6 glycosidic bonds.
 15. Thecomposition of claim 12, wherein the glucan has an average molecularweight of about 15 to about 350 kD.
 16. The composition of claim 12,wherein the glucan is a particulate or soluble glucan.
 17. Thecomposition of claim 12, wherein the composition is formulated for oraladministration.
 18. A method for delivering an isolated DNA, RNA orplasmid into cells, comprising administering to said cells a compositioncomprising said DNA, RNA or plasmid and an effective amount of a yeastβ-glucan, wherein said β-glucan is not encapsulated.
 19. The method ofclaim 18, wherein the glucan comprises β-1,3 bonds in the backbone. 20.The method of claim 19, wherein the glucan further comprises branches ofβ-1,3-linked glucose units, attached to the backbone via β-1,6glycosidic bonds.
 21. The method of claim 18, wherein the glucan has anaverage molecular weight of about 15 to about 350 kD.
 22. The method ofclaim 18, wherein the glucan is a particulate or soluble glucan.
 23. Themethod of claim 18, wherein the composition is orally administered.